CN107429298B - Method for designing primer for polymerase chain reaction and primer combination - Google Patents

Method for designing primer for polymerase chain reaction and primer combination Download PDF

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CN107429298B
CN107429298B CN201680018913.3A CN201680018913A CN107429298B CN 107429298 B CN107429298 B CN 107429298B CN 201680018913 A CN201680018913 A CN 201680018913A CN 107429298 B CN107429298 B CN 107429298B
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辻本尧之
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Abstract

The present invention provides a method for designing a primer for polymerase chain reaction and a primer combination, the method comprising: a local alignment step of obtaining a local alignment score by locally aligning the base sequences of the primer candidates with the double sequences under such a condition that a part of the sequences to be compared include the 3' -end of the base sequence of the primer candidate; and a global alignment step of performing global alignment of base sequences of a predetermined sequence length including the 3' -end of the base sequence of the primer candidate in a double sequence to obtain a global alignment score.

Description

Method for designing primer for polymerase chain reaction and primer combination
Technical Field
The invention relates to a design method of a primer for polymerase chain reaction and a primer combination.
Background
In recent years, the popularization of next-generation sequencing technology has made it easier to guarantee the quality and quantity of base sequence data, and therefore gene analysis can be performed easily. Introduction of NGS (Next Generation Sequencing) technology has gradually eliminated most of the technical difficulties of genome-wide analysis. However, in the case of the human genome, the total base length of the genome is usually 30 hundred million base pairs or more, which is large, and even when the NGS technique is used to perform genome-wide analysis, considerable cost and time are required.
On the other hand, it cannot be said that the whole genome analysis is most suitable as a method for achieving the purpose of detecting gene abnormality. This is because it is sufficient to analyze only a gene region associated with a gene abnormality. The gene region associated with a gene abnormality includes both a coding region and a non-coding region. Therefore, PCR (Polymerase Chain Reaction) is widely used as a technique for efficiently and highly accurately performing gene analysis by amplifying only a desired specific gene region and restricting the region to the nucleotide sequence for reading. In particular, a method of selectively amplifying a plurality of gene regions by simultaneously supplying a plurality of primers to a certain PCR reaction system is called multiplex PCR.
However, generally, the number of regions simultaneously amplified by multiplex PCR cannot be set to be too large. One of the reasons for this is that the primers react with each other to produce an extra amplification product called a primer dimer, which makes it impossible to efficiently amplify the target gene region. This problem is significant when the amount of DNA (deoxyribose nucleic acid) that becomes a template for PCR reaction, such as single cell (single cell) analysis, is extremely small.
As a method for suppressing the formation of a primer dimer, for example, patent document 1 describes a method in which a base sequence of a primer is divided into a stable region and a variable region, the same base sequence is arranged in the stable region, and only 2 bases, such as those of cytosine (C), thymidine (T), guanine (G), and adenine (a), which are not complementary to each other, are defined in the variable region, thereby applying PCR to a large number of regions. Further, patent document 2 describes that, for all combinations of primers, a score indicating complementarity at the 3 '-ends between the primers (a local alignment score at the 3' -ends) is calculated, and a combination of primers having low complementarity between the primers is selected, thereby reducing the possibility that different primers of interest form primer dimers with each other by multiplex PCR.
Prior art documents
Patent document
Patent document 1: international publication No. 2004/081225
Patent document 2: international publication No. 2008/004691
Disclosure of Invention
Technical problem to be solved by the invention
As a result of studies by the present inventors, the method described in patent document 1 aims to provide a method for amplifying a whole genome region without bias by providing a universal primer, and only a region including a base sequence of a specific variable region can be targeted, and thus it is not possible to efficiently select only a plurality of specific regions. Further, in a PCR reaction with a very small amount of template DNA such as multiplex PCR from a single cell, it is also important to consider a primer dimer formed by annealing only the ends of the primers, but the method described in patent document 2 does not take this into account. As a result, the methods described in patent documents 1 and 2 cannot efficiently selectively amplify only a plurality of regions on the genome at a level required in recent years.
Accordingly, an object of the present invention is to provide a method for designing a primer for polymerase chain reaction, which can selectively and efficiently amplify a target gene region.
Means for solving the technical problem
The present inventors have made intensive studies to solve the above problems, and as a result, have found the following and completed the present invention: the primer dimer formation is evaluated, and a primer set for polymerase chain reaction, which can selectively and efficiently amplify a target gene region, can be obtained by performing local alignment of base sequences of primer candidates in two sequences under the condition that a part of the sequences to be compared include the 3 '-end of the base sequence of the primer, obtaining a local alignment score, performing stage 1 selection based on the obtained local alignment score, performing global alignment of base sequences having a preset sequence length including the 3' -end of the base sequence of the primer candidates in two sequences, obtaining a global alignment score, performing stage 2 selection based on the obtained global alignment score, and using primers selected in any of stage 1 and stage 2.
That is, the present invention is (1) to (3) below.
(1) A method for designing a primer for polymerase chain reaction, comprising:
a target region selection step of selecting a target region amplified by the polymerase chain reaction from a region on a genome;
a primer candidate base sequence creating step of creating at least 1 base sequence of each primer candidate for amplifying the target region, based on the base sequences of the regions in the vicinity of both ends of the target region on the genome;
a local alignment step of obtaining a local alignment score by locally aligning the base sequences of the primer candidates with respect to the double sequences under a condition that a part of the sequences to be compared include the 3' -end of the base sequence of the primer candidate;
a stage 1 selection step of performing stage 1 selection of the base sequence of the primer candidate based on the local alignment score obtained in the local alignment step;
a global alignment step of obtaining a global alignment score by globally aligning base sequences of a predetermined sequence length including the 3' -end of the base sequence of the primer candidate in a double sequence;
a stage 2 selection step of selecting stage 2 of the base sequences of the primer candidates based on the global alignment score obtained in the global alignment step; and
a primer-using step of using, as a base sequence of a primer for amplifying the target region, a base sequence of a candidate primer selected in either of the step 1 and the step 2,
wherein the two steps of the local alignment step and the stage 1 selection step are performed before or after the two steps of the global alignment step and the stage 2 selection step, or are performed simultaneously with the two steps of the global alignment step and the stage 2 selection step.
(2) The method for designing a primer for polymerase chain reaction according to (1) above, comprising:
a 1 st target region selection step of selecting a 1 st target region amplified by the polymerase chain reaction from a region on a genome;
a 1 st primer candidate base sequence preparing step of preparing at least 1 base sequence of each primer candidate for amplifying the 1 st target region, based on the base sequences of the respective vicinal regions at both ends of the 1 st target region on the genome;
a 1 st local alignment step of obtaining a local alignment score by performing local alignment of double sequences on the base sequences of the primer candidates under such a condition that a part of the sequences to be compared include the 3' -end of the base sequence of the primer candidate;
a first stage 1 selection step of performing stage 1 selection of the base sequences of the primer candidates based on the local alignment score obtained in the local alignment step;
a 1 st global alignment step of performing global alignment of base sequences of a predetermined sequence length including the 3' -end of the base sequence of the primer candidate in a double sequence to obtain a global alignment score;
a first 2 nd stage selection step of selecting the 2 nd stage of the base sequence of the primer candidate based on the global alignment score obtained in the global alignment step;
a 1 st primer application step of applying, as a base sequence of a primer for amplifying the 1 st target region, a base sequence of a candidate primer selected in either of the first 1 st and 2 nd step;
a 2 nd target region selection step of selecting a 2 nd target region amplified by the polymerase chain reaction from a region on the genome;
a 2 nd primer candidate base sequence preparing step of preparing at least 1 base sequence of each primer candidate for amplifying the 2 nd target region, based on the base sequences of the regions in the vicinity of both ends of the 2 nd target region on the genome;
a 2 nd local alignment step of obtaining a local alignment score by locally aligning the nucleotide sequence of the primer candidate for amplifying the 2 nd target region and the nucleotide sequence of the used primer in a double sequence under a condition that a part of the sequences to be compared includes the nucleotide sequence of the primer candidate and the 3' -end of the nucleotide sequence of the used primer;
a second stage 1 selection step of performing stage 1 selection of a base sequence of a candidate primer for amplifying the 2 nd target region based on the local alignment score;
a 2 nd global alignment step of obtaining a global alignment score by globally aligning base sequences of a predetermined sequence length including the base sequence of the primer candidate for amplifying the 2 nd target region and the 3' -end of the base sequence of the used primer in a double sequence;
a second stage 2 selection step of performing stage 2 selection of a base sequence of a candidate primer for amplifying the 2 nd target region based on the global alignment score; and
a 2 nd primer-employing step of employing, as a base sequence of a primer for amplifying the 2 nd target region, a base sequence of a candidate primer selected in either of the second 1 st-stage selection step and the second 2 nd-stage selection step,
wherein the 1 st local alignment step and the first 1 st stage selection step are performed before or after the 1 st global alignment step and the first 2 nd stage selection step, or simultaneously with the 1 st global alignment step and the first 2 nd stage selection step, and
the two steps of the 2 nd local alignment step and the second 1 st stage selection step are performed before or after the two steps of the 2 nd global alignment step and the second 2 nd stage selection step, or simultaneously with the two steps of the 2 nd global alignment step and the second 2 nd stage selection step,
when the target region is 3 or more, the steps from the 2 nd target region selection step to the 2 nd primer application step are repeated for all target regions until the base sequence of the primer for amplifying the target region is applied.
(3) A primer combination for polymerase chain reaction, wherein,
the nucleotide sequences of the primers are partially aligned in duplicate under the condition that the partial sequences to be compared include the 3' -end of the nucleotide sequence, and the obtained partial alignment score is less than the 1 st threshold value
The nucleotide sequences including the predetermined number of nucleotides at the 3' -end of the nucleotide sequence of each primer are globally aligned in a double sequence, and the obtained global alignment score is less than the 2 nd threshold.
Effects of the invention
According to the present invention, a method for designing a primer for polymerase chain reaction that can selectively and efficiently amplify a target gene region can be provided.
Further, the present invention can provide a primer set for polymerase chain reaction that can selectively and efficiently amplify a target gene region.
Drawings
FIG. 1 is a block diagram of a method for designing a primer of the present invention.
FIG. 2 is a diagram showing a partial alignment of a pair of the base sequence represented by SEQ ID No. 1 and the base sequence represented by SEQ ID No. 2 in example 1.
FIG. 3 is a diagram showing a partial alignment of a pair of the base sequence represented by SEQ ID No. 1 and the base sequence represented by SEQ ID No. 3 in example 1.
FIG. 4 is a diagram showing a partial alignment of a pair of the base sequence represented by SEQ ID No. 1 and the base sequence represented by SEQ ID No. 4 in example 1.
FIG. 5 is a diagram showing a partial alignment of a pair of the base sequence represented by SEQ ID No. 1 and the base sequence represented by SEQ ID No. 5 in example 1.
FIG. 6 is a diagram showing a partial alignment of a pair of the base sequence represented by SEQ ID No. 1 and the base sequence represented by SEQ ID No. 6 in example 1.
FIG. 7 is a diagram showing a partial alignment of a pair of the base sequence represented by SEQ ID No. 1 and the base sequence represented by SEQ ID No. 7 in example 1.
FIG. 8 is a diagram showing a partial alignment of a pair of the base sequence represented by SEQ ID No. 1 and the base sequence represented by SEQ ID No. 8 in example 1.
FIG. 9 is a diagram showing a partial alignment of a pair of the base sequence represented by SEQ ID No. 2 and the base sequence represented by SEQ ID No. 3 in example 1.
FIG. 10 is a diagram showing a partial alignment of a pair of the base sequence represented by SEQ ID No. 2 and the base sequence represented by SEQ ID No. 4 in example 1.
FIG. 11 is a diagram showing a partial alignment of a pair of the base sequence represented by SEQ ID No. 2 and the base sequence represented by SEQ ID No. 5 in example 1.
FIG. 12 is a diagram showing a partial alignment of a pair of the base sequence represented by SEQ ID No. 2 and the base sequence represented by SEQ ID No. 6 in example 1.
FIG. 13 is a diagram showing a partial alignment of a pair of the base sequence represented by SEQ ID No. 2 and the base sequence represented by SEQ ID No. 7 in example 1.
FIG. 14 is a diagram showing a partial alignment of a pair of the base sequence represented by SEQ ID No. 2 and the base sequence represented by SEQ ID No. 8 in example 1.
FIG. 15 is a diagram showing a partial alignment of a pair of the base sequence represented by SEQ ID No. 3 and the base sequence represented by SEQ ID No. 4 in example 1.
FIG. 16 is a diagram showing a partial alignment of a pair of the base sequence represented by SEQ ID No. 3 and the base sequence represented by SEQ ID No. 5 in example 1.
FIG. 17 is a diagram showing a partial alignment of a pair of the base sequence represented by SEQ ID No. 3 and the base sequence represented by SEQ ID No. 6 in example 1.
FIG. 18 is a diagram showing a partial alignment of a pair of the base sequence represented by SEQ ID No. 3 and the base sequence represented by SEQ ID No. 7 in example 1.
FIG. 19 is a diagram showing a partial alignment of a pair of the base sequence represented by SEQ ID No. 3 and the base sequence represented by SEQ ID No. 8 in example 1.
FIG. 20 is a diagram showing a partial alignment of a pair of the base sequence represented by SEQ ID No. 4 and the base sequence represented by SEQ ID No. 5 in example 1.
FIG. 21 is a diagram showing a partial alignment of a pair of the base sequence represented by SEQ ID No. 4 and the base sequence represented by SEQ ID No. 6 in example 1.
FIG. 22 is a drawing showing a partial alignment of a pair of the base sequence represented by SEQ ID No. 4 and the base sequence represented by SEQ ID No. 7 in example 1.
FIG. 23 is a diagram showing a partial alignment of a pair of the base sequence represented by SEQ ID No. 4 and the base sequence represented by SEQ ID No. 8 in example 1.
FIG. 24 is a diagram showing a partial alignment of a pair of the base sequence represented by SEQ ID No. 5 and the base sequence represented by SEQ ID No. 6 in example 1.
FIG. 25 is a diagram showing a partial alignment of a pair of the base sequence represented by SEQ ID No. 5 and the base sequence represented by SEQ ID No. 7 in example 1.
FIG. 26 is a diagram showing a partial alignment of a pair of the base sequence represented by SEQ ID No. 5 and the base sequence represented by SEQ ID No. 8 in example 1.
FIG. 27 is a diagram showing a partial alignment of a pair of the base sequence represented by SEQ ID No. 6 and the base sequence represented by SEQ ID No. 7 in example 1.
FIG. 28 is a drawing showing a partial alignment of a pair of the base sequence represented by SEQ ID No. 6 and the base sequence represented by SEQ ID No. 8 in example 1.
FIG. 29 is a drawing showing a partial alignment of a pair of the base sequence represented by SEQ ID No. 7 and the base sequence represented by SEQ ID No. 8 in example 1.
FIG. 30 is a drawing showing global alignments of 2 bases at the 3' -end of a pair of arbitrary 2 base sequences selected from the base sequences shown in SEQ ID Nos. 1 to 8 in example 1.
FIG. 31 is a drawing showing a partial alignment of a pair of the base sequence represented by SEQ ID No. 9 and the base sequence represented by SEQ ID No. 10 in comparative example 1.
FIG. 32 is a drawing showing a partial alignment of a pair of the base sequence represented by SEQ ID No. 11 and the base sequence represented by SEQ ID No. 12 in comparative example 2.
FIG. 33 is a diagram showing a global alignment of 2 bases at the 3' -end of a pair of a base sequence represented by SEQ ID No. 11 and a base sequence represented by SEQ ID No. 12 in comparative example 2.
Detailed Description
First, the points of the present invention advantageous over the prior art will be described.
The present invention is advantageous over the conventional technique described in patent document 1 in that the technique described in patent document 1 is intended to provide a non-biased amplification method for the whole genome region by providing a universal primer, and is not intended to selectively amplify a specific gene region, but the present invention is capable of selectively and efficiently amplifying a target gene region. Further, as an advantageous point of the present invention over the conventional technology described in patent document 2, there is an aspect in which the technology described in patent document 2 is a technology in which a primer combination in which a primer dimer is not easily formed is designed by attempting to select a primer having low complementarity of the entire sequence by local alignment of the entire primer base sequence, but the formation of a primer dimer cannot be sufficiently prevented only by reducing the complementarity of the entire sequence, whereas in the present invention, the complementarity of the entire sequence including the 3 'end is reduced by local alignment, and a primer set is prepared by global alignment so as to reduce the complementarity of a partial sequence having an extremely short length of, for example, about 5 nucleotides or less at the 3' end, so that a target gene region can be selectively and efficiently amplified.
The present invention will be described in detail below.
[ method of designing primer for polymerase chain reaction (embodiment 1) ]
The 1 st embodiment of the method for designing a primer for polymerase chain reaction of the present invention is a method for designing a primer for polymerase chain reaction, comprising:
(a) a target region selection step of selecting a target region amplified by the polymerase chain reaction from a region on a genome;
(b) a primer candidate base sequence creating step of creating at least 1 base sequence of each primer candidate for amplifying the target region, based on the base sequences of the regions in the vicinity of both ends of the target region on the genome;
(c) a local alignment step of obtaining a local alignment score by locally aligning the base sequences of the primer candidates with respect to the double sequences under a condition that a part of the sequences to be compared include the 3' -end of the base sequence of the primer candidate;
(d) a stage 1 selection step of performing stage 1 selection of the base sequences of the primer candidates based on the local alignment score obtained in the local alignment step (c);
(e) a global alignment step of obtaining a global alignment score by globally aligning base sequences of a predetermined sequence length including the 3' -end of the base sequence of the primer candidate in a double sequence;
(f) a 2 nd-stage selection step of performing 2 nd-stage selection of the base sequences of the primer candidates based on the global alignment score obtained in the (e) global alignment step; and
(g) a primer-using step of using, as a base sequence of a primer for amplifying the target region, a base sequence of a candidate primer selected in both the step (d) of selecting at the 1 st stage and the step (f) of selecting at the 2 nd stage,
wherein the two steps of the local alignment step and the stage 1 selection step are performed before or after the two steps of the global alignment step and the stage 2 selection step, or are performed simultaneously with the two steps of the global alignment step and the stage 2 selection step.
The respective steps of embodiment 1 of the method for designing a primer for polymerase chain reaction of the present invention will be described in detail.
(a) Target region selection step
Shown in the block diagram of fig. 1 as "(1 st target region selection process").
The target region selection step is a step of selecting a target region amplified by polymerase chain reaction from a region on the genome.
(regions on the genome)
In the present invention, the "region on the genome" refers to a region on the genomic DNA where a site related to a gene polymorphism, a monogenic disease, a multifactorial disease, cancer, or the like exists. The length of the region is not particularly limited, and may be 1 base or more.
The region on the genome from which the target region is selected may be present in any one of the gene region and the nongenic region. The gene region includes a coding region in which a gene encoding a protein, a ribosomal RNA (Ribonucleic acid) gene, a transfer RNA gene, and the like are present, and a non-coding region in which an intron of a gene is divided, a transfer regulatory region, a 5 '-leader sequence, a 3' -trailer sequence, and the like are present. The nongenic region includes a non-repetitive sequence such as a pseudogene, a spacer, a response element, and an origin of replication, and a repetitive sequence such as a tandem repetitive sequence and a dispersed repetitive sequence.
Examples of the gene Polymorphism include SNP (Single Nucleotide Polymorphism), SNV (Single Nucleotide variation), STRP (Short derived Repeat Polymorphism), mutation, and insertion and/or deletion (indel).
A monogenic disease is a disease that develops as a result of a single gene abnormality. Examples of the abnormality include deletion or duplication of the gene itself and/or substitution, insertion and/or deletion of a base in the gene. A single gene causing a single gene disease is referred to as a "disease gene".
The multifactorial disease is a disease in which a plurality of genes intervene, and may be associated with a specific combination of SNPs or the like. These genes are referred to as "susceptibility genes" in view of the meaning of having a susceptibility to a disease.
Cancer is a disease caused by genetic variation. As with other diseases, hereditary (familial) cancers, called hereditary tumors (familial tumors), and the like, also exist among cancers.
The number of regions on the genome is not particularly limited. This is because a region on the genome is a candidate list for selecting a target region, and even if there are a large number of lists, it is not necessary to design primers for all the lists.
(target region)
The target region is a region selected from the above-mentioned regions on the genome as targets for amplification by polymerase chain reaction. The purpose of selection is not limited to detection of gene polymorphisms, diseases, cancers, and the like associated with each region, and may be detection of chromosomal aneuploidy and the like. The purpose of selection is not limited to 1, and may be 2 or more.
The number of regions on the genome selected as the target region varies depending on the purpose, but is not particularly limited as long as it is 1 or more, and is usually preferably 3 or more, more preferably 5 or more, and still more preferably 10 or more.
(polymerase chain reaction)
In the present invention, Polymerase Chain Reaction (PCR) is a reaction for synthesizing DNA from a template DNA using DNA polymerase. Unlike DNA synthesis in cells, PCR requires 1 or more kinds of oligonucleotides called primers, and usually 2 or more kinds, for synthesizing DNA. The combination of primers used simultaneously in 1 PCR reaction system is sometimes referred to as a primer combination.
PCR can be easily extended from a simple system in which 1 region is amplified using 1 pair of primer combination to a complex system in which a plurality of regions are simultaneously amplified using a plurality of pairs of primer combinations (multiplex PCR).
PCR has the advantage of being able to selectively amplify only desired regions from very large DNA molecules, such as the human genome (30 hundred million base pairs). In addition, a sufficient amount of amplification products of a desired region can be obtained using a very small amount of genomic DNA as a template.
The advantages of PCR vary depending on the protocol, but the time required for amplification is about 2 hours, which is short.
Further, PCR has advantages in that it can be amplified by a fully automated desktop facility with a simple process.
(b) Process for preparing candidate base sequence of primer
Shown in the block diagram of FIG. 1 are "(1 st step of preparing a candidate base sequence for a primer").
The primer candidate base sequence creating step is a step of creating at least 1 base sequence of each primer candidate for amplifying the target region, based on the base sequences of the regions in the vicinity of both ends of the target region on the genome.
The vicinity of the target region is a general term for the region outside the 5 'end of the target region and the region outside the 3' end of the target region. The inner side of the target region is not included in the nearby region.
The length of the region in the vicinity is not particularly limited, but is preferably not more than a length that can be extended by PCR, and more preferably not more than the upper limit of the length of the DNA fragment to be amplified. In particular, it is preferred that the length of the concentrated selection and/or sequence reads be easily implemented. The type of the enzyme (DNA polymerase) used in PCR may be appropriately changed depending on the type thereof. Specifically, the length of the region in the vicinity is preferably about 20 to 500 bases, more preferably about 20 to 300 bases, still more preferably about 20 to 200 bases, and yet more preferably about 50 to 200 bases.
In addition, the base sequences of the primer candidates are prepared in the same manner as those noted in the ordinary primer design method, such as the length of the primer, the GC content (which indicates the total mole percentage of guanine (G) and cytosine (C) in all nucleic acid bases), the Tm value (which is the temperature at which 50% of double-stranded DNA is dissociated into single-stranded DNA, Tm is derived from the long temperature), and the sequence deviation.
The length (number of nucleotides) of the primer is not particularly limited, but is preferably 15mer to 45mer, more preferably 15mer to 35mer, still more preferably 15mer to 25mer, and still more preferably 15mer to 20 mer. When the length of the primer is within this range, it is easy to design a primer having excellent characteristics and amplification efficiency.
The GC content is not particularly limited, but is preferably 40 to 60 mol%, more preferably 45 to 55 mol%. When the GC content is within this range, the problems of deterioration in properties and amplification efficiency due to higher-order structures are unlikely to occur.
The Tm value is not particularly limited, but is preferably within a range of 50 to 65 ℃ and more preferably within a range of 55 to 65 ℃.
The Tm value can be calculated by Software such as OLIGO Primer Analysis Software (manufactured by Molecular Biology instruments) or Primer3(http:// www-genome. wi. mit. edu/ftp/distribution/Software /).
Further, the number of A, T, G and C (nA, nT, nG, and nC, respectively) in the base sequence of the primer can be determined by calculation from the following formula.
Tm (. degree. C.). gtoreq.2 (nA + nT) +4(nC + nG)
The method of calculating the Tm value is not limited to this, and the Tm value can be calculated by various conventionally known methods.
The nucleotide sequences of the primer candidates are preferably sequences having no base bias as a whole. For example, local GC-rich sequences and local AT-rich sequences are preferably avoided.
Furthermore, T and/or C continuations (polypyrimidines) and a and/or G continuations (polypurines) are preferably also avoided.
Furthermore, it is preferable that the 3' -end base sequence avoid GC-rich sequences or AT-rich sequences. The 3' -terminal base is preferably G or C, but not limited thereto.
(characteristic inspection step)
If necessary, a characteristic inspection step of evaluating the characteristics of the base sequences of the primer candidates based on the sequence complementarity of the base sequences of the primer candidates prepared in the above-mentioned (b) primer candidate base sequence preparation step with respect to the respective genomic DNAs.
In the examination of the properties, the base sequence of the genomic DNA and the base sequence of the primer candidate are locally aligned, and when the local alignment score is less than a predetermined value, it can be evaluated that the complementarity of the base sequence of the primer candidate with respect to the genomic DNA is low and the properties are high. Among them, it is preferable to perform local alignment of complementary strands of genomic DNA. This is because the primer is a single-stranded DNA, and the genomic DNA is double-stranded. In addition, a nucleotide sequence complementary to the candidate nucleotide sequence of the primer may be used instead of the candidate nucleotide sequence of the primer. Complementarity can be considered as homology with respect to the complementary strand.
Further, a homology search may be performed on a genomic DNA base sequence database using the base sequences of the primer candidates as query sequences. Examples of homology Search tools include BLAST (Basic Local Alignment Search Tool) (Altschul, S.A., top 4, Basic Local Alignment Search Tool ", Journal of Molecular Biology, 1990, 10 months, 215 th, p.403-410), and FASTA (Pearson, W.R., top 1, Improved tools for biological sequence composition), American college of sciences, 1988, 4 months, 85 th, p.2444-2448). As a result of performing a homology search, a local alignment can be obtained.
The scoring system and the threshold for the local alignment scoring are not particularly limited, and can be appropriately set according to the length of the base sequence of the primer candidate, the PCR conditions, and the like. When using a homology search tool, a default value for the homology search tool may be used.
For example, a scoring system may be considered in which a complementary base (match) ═ 1, a non-complementary base (mismatch) ═ 1, and an insertion and/or deletion (gap penalty) — 3 are used, and the threshold is set to + 15.
When the nucleotide sequence of the primer candidate is complementary to the nucleotide sequence of the predicted position on the genomic DNA and has low characteristics, the primer candidate may be excluded because artifacts other than the target region are amplified when PCR is performed using the primer having the nucleotide sequence.
(c) Local alignment procedure
Shown in the block diagram of fig. 1 as "(1 st partial alignment step").
The local alignment process comprises the following steps: the local alignment score is obtained by performing local alignment of the duplexes under the condition that a part of the sequences to be compared include the 3' -end of the base sequence, for all pairs including 2 base sequences extracted from the base sequences of the primer candidates for amplifying the target region prepared in the step (b) of preparing the base sequences of the primer candidates.
The combination of pairs of base sequences to be locally aligned may be a combination selected to allow duplication or a combination selected to disallow duplication, but when primer dimer formation between primers having the same base sequence has not been evaluated, it is preferable to use a combination selected to allow duplication.
Regarding the total number of combinations, the number of nucleotide sequences prepared in the step of preparing a candidate nucleotide sequence of the primer (b) is m, and when the nucleotide sequences are selected while allowing the nucleotide sequences to repeat, the number of combinations is "mH2＝m+1C2(m + 1)! 2 (m-1)! ", when selected without allowing repetition, is"mC2＝m(m-1)/2”。
When the global alignment step (e) and the 2 nd-stage selection step (f) described later are performed first, the present step and the 1 st-stage selection step (d) described later can be performed on the primer candidates selected in the 2 nd-stage selection step (f).
Local alignment is alignment of a part of a sequence, and a part having high complementarity can be examined locally.
However, in the present invention, unlike the local alignment of the base sequences which is usually performed, the local alignment is performed under such a condition that "a part of the sequences to be compared includes the 3 'ends of the base sequences" so that the part of the sequences to be compared includes the 3' ends of both the base sequences. In the present invention, the following is preferred: under the condition of "the partial sequences to be compared include the 3 '-ends of the nucleotide sequences", that is, "alignment considering only the partial sequences to be compared starting from the 3' -end of one of the sequences and ending at the 3 '-end of the other sequence", the partial alignments are performed so that the partial sequences to be compared include the 3' -ends of both the nucleotide sequences.
In addition, local alignments can be inserted into the gap. Gaps represent insertions and/or deletions (indels) of bases.
In the local alignment, the case of complementarity between the base sequence pairs is regarded as matching (matching), and the case of non-complementarity is regarded as non-matching (mismatching).
The alignment is performed in such a manner that scores are given to match, mismatch, and insertion and/or deletion, respectively, and the total score is maximized. The score can be set appropriately. For example, the scoring system may be set as shown in table 1 below. In addition, "-" in table 1 indicates a gap (insertion and/or deletion (indel)).
[ Table 1]
TABLE 1
Figure BDA0001420562140000141
For example, it is considered that the nucleotide sequences of SEQ ID Nos. 1 and 2 shown in Table 2 below are partially aligned. The scoring system is shown in table 1.
[ Table 2]
TABLE 2
Figure BDA0001420562140000142
Dot matrices (Dot matrices) shown in Table 3 were prepared based on the nucleotide sequences of SEQ ID Nos. 1 and 2. Specifically, the base sequence of SEQ ID No. 1 was aligned from left to right in the 5 'to 3' direction, the base sequence of SEQ ID No. 2 was aligned from bottom to top in the 5 'to 3' direction, and a lattice having base complementarity was represented by ". cndot", thereby obtaining the lattice shown in Table 3.
[ Table 3]
TABLE 3
Figure BDA0001420562140000151
An alignment (double-sequence alignment) of a part of the sequences shown in Table 4 below was obtained from the lattice shown in Table 3 (see the shaded part in Table 3).
[ Table 4]
TABLE 4
Figure BDA0001420562140000152
The local alignment score is "-4" due to matches (+1) × 2, mismatches (-1) × 6, insertions and/or deletions (-3) × 0.
The alignment (double sequence alignment) is not limited to the lattice method exemplified herein, and can be obtained by a dynamic programming method, a lexical method, or other various methods.
(d) Stage 1 selection procedure
Shown in the block diagram of fig. 1 as "(first) stage 1 election process".
The step of selecting at stage 1 is a step of selecting at stage 1 the base sequence of the primer candidate created in the step of creating a base sequence candidate for the primer created in the step of (b) based on the local alignment score obtained in the step of (c).
The threshold value (1 st threshold value) of the local alignment score is set in advance.
If the local alignment score is less than the 1 st threshold, it is determined that the dimer formation of the 2 base sequence pairs is low, and the subsequent steps are performed. On the other hand, if the local alignment score is not less than the 1 st threshold, it is determined that the dimer formation of the pair of 2 base sequences is high, and the subsequent step is not performed on the pair.
The 1 st threshold is not particularly limited and can be set as appropriate. For example, the 1 st threshold may be set according to PCR conditions such as the amount of genomic DNA that becomes a template for polymerase chain reaction.
Here, a case where the 1 st threshold is set to "3" in the example shown in the local alignment step (c) is considered.
In the above example, since the local alignment score was "-3" and was smaller than "3" which is the 1 st threshold, it was judged that the dimer formation property of the pair of base sequences of SEQ ID Nos. 1 and 2 was low.
In addition, this step is performed for all pairs for which scores are calculated in the local alignment step (c).
(e) Global alignment procedure
Shown in the block diagram of fig. 1 as "(1 st) global alignment procedure".
The global alignment step is a step of obtaining a global alignment score by globally aligning, in a double sequence, all pairs including 2 nucleotide sequences extracted from the nucleotide sequences of the primer candidates for amplifying the target region prepared in the primer candidate nucleotide sequence preparation step (b) with nucleotide sequences including the nucleotide sequence of the primer candidate at the 3' -end of the nucleotide sequence of a predetermined sequence length.
The combination of pairs of base sequences to be globally aligned may be a combination selected to allow duplication or a combination selected not to allow duplication, but when primer dimer formation between primers of the same base sequence has not been evaluated, a combination selected to allow duplication is preferable.
Regarding the total number of combinations, the number of nucleotide sequences prepared in the step of preparing a candidate nucleotide sequence of the primer (b) is m, and when the nucleotide sequences are selected while allowing the nucleotide sequences to repeat, the number of combinations is "mH2＝m+1C2(m + 1)! 2 (m-1)! ", when selected without allowing repetition, is"mC2＝m(m-1)/2”。
When the above-mentioned two steps of (c) local alignment step and (d) selection step at stage 1 are carried out first, this step and the later-described selection step at stage 2 (f) can be carried out on the primer candidates selected in the selection step at stage 1 (d).
Global alignment is an alignment made to "the entire sequence", which can be checked for complementarity.
However, the "entire sequence" herein refers to the entire nucleotide sequence having a predetermined sequence length including the 3' -end of the nucleotide sequence of the primer candidate.
In addition, global alignments can be inserted into gaps. Gaps represent insertions and/or deletions (indels) of bases.
In the global alignment, the case of complementarity between the base sequence pairs is regarded as matching (matching), and the case of non-complementarity is regarded as non-matching (mismatching).
The alignment is performed in such a manner that scores are given to match, mismatch, and insertion and/or deletion, respectively, and the total score is maximized. The score can be set appropriately. For example, the scoring system may be set as shown in table 1. In table 1, "-" indicates a gap (insertion and/or deletion (indel)).
For example, it is considered that 3 bases (capital letters. correspond to "base sequences having a predetermined sequence length including a 3 'end") at each 3' end are globally aligned with the base sequences of SEQ ID Nos. 1 and 2 shown in Table 5 below. The scoring system is shown in table 1.
[ Table 5]
TABLE 5
Figure BDA0001420562140000171
When 3 bases (capital letter parts) at the 3 'end of the base sequence of SEQ ID No. 1 and 3 bases (capital letter parts) at the 3' end of SEQ ID No. 2 are aligned globally so that the scores are maximized, alignments (double sequence alignments) shown in Table 6 below can be obtained.
[ Table 6]
TABLE 6
Figure BDA0001420562140000172
The global alignment score is "-3" due to matches (+1) × 0, mismatches (-1) × 3, insertions and/or deletions (-3) × 0.
In addition, alignment (two sequence alignment) can be achieved by lattice method, dynamic programming method, lexical method or other various methods.
(f) Stage 2 selection procedure
Shown in the block diagram of fig. 1 as "(first) stage 2 election process".
The 2 nd-stage selection step is a step of performing 2 nd-stage selection of the base sequence of the primer candidate created in the (b) primer candidate base sequence creating step, based on the global alignment score obtained in the (e) global alignment step.
The threshold value (2 nd threshold value) of the global alignment score is set in advance.
If the global alignment score is less than the 2 nd threshold, it is determined that the dimer formation of the 2 base sequence pairs is low, and the subsequent steps are performed. On the other hand, if the global alignment score is not less than the 2 nd threshold, it is determined that the dimer formation of the pair of 2 base sequences is high, and the subsequent steps are not performed on the pair.
The 2 nd threshold is not particularly limited and can be set as appropriate. For example, the 2 nd threshold may be set according to PCR conditions such as the amount of genomic DNA that becomes a template for polymerase chain reaction.
In addition, the global alignment score obtained by globally aligning base sequences of a predetermined number of bases including the 3 '-end of the base sequence of each primer in a double sequence can be set to be less than the 2 nd threshold by setting the base sequences of several bases up to the 3' -end of the primer as the same base sequence.
Here, a case where the 2 nd threshold is set to "3" in the example shown in the above (e) global alignment step is considered.
In the above example, since the global alignment score was "-3" and was smaller than "3" which is the 2 nd threshold, it could be judged that the dimer formation property of the pair of base sequences of SEQ ID Nos. 1 and 2 was low.
In addition, this step is performed for all pairs for which scores are calculated in the global alignment step (e).
The two steps of (c) the local alignment step and (d) the 1 st-stage selection step may be performed before or after the two steps of (e) the global alignment step and (f) the 2 nd-stage selection step, or may be performed simultaneously with the two steps of (e) the global alignment step and (f) the 2 nd-stage selection step.
In order to reduce the amount of calculation, it is preferable that the global alignment step (e) and the 2 nd-stage selection step (f) are performed first, and the local alignment step (c) and the 1 st-stage selection step (d) are performed in combination in the 2 nd-stage selection step (f). In particular, the effect of reducing the amount of calculation increases as the number of target regions increases and the number of base sequences of primer candidates increases, and the entire process can be speeded up.
This is because, since the base sequences having a short length such as "a predetermined sequence length" are aligned globally in the global alignment step (e), the calculation amount is less than the local alignment score for finding a partial sequence having high complementarity from the entire base sequences under the condition including the 3' -end, and the processing can be performed quickly. In addition, in a commonly known algorithm, it is known that the global alignment speed is faster than the local alignment speed when the sequences having the same length are aligned.
(step of examining the length of amplification sequence)
If necessary, an amplified sequence length check step of calculating the distance between the ends of the base sequences of the primer candidates on the genomic DNA or chromosomal DNA for the pair of base sequences of the primer candidates determined to have low primer dimer formation in the above-mentioned (d) stage 1 selection step and the above-mentioned (f) stage 2 selection step, and determining whether the distance is within a predetermined range may be performed.
When the distance between the ends of the base sequences is within a predetermined range, it is highly likely that the pair of base sequences that are candidates for the primer can appropriately amplify the target region. The distance between the ends of the nucleotide sequences of the primer candidates is not particularly limited, and can be set appropriately according to PCR conditions such as the type of enzyme (DNA polymerase). For example, the number of the base pairs may be set to various ranges such as 100 to 200 base pairs, 120 to 180 base pairs, 140 to 160 base pairs, and 160 to 180 base pairs.
(g) Procedure for primer application
Shown in the block diagram of FIG. 1 as "(1 st primer application step").
The primer-employing step is a step of employing, as a base sequence of a primer for amplifying the target region, a base sequence of a candidate primer selected in both the step (d) of selecting at the 1 st stage and the step (f) of selecting at the 2 nd stage.
That is, in this step, the base sequences of the primer candidates are used as the base sequences of the primers for amplifying the target region, and the base sequences of the primer candidates are subjected to local alignment under the condition that a part of the sequences to be compared include the 3 '-end of the base sequence, and the local alignment score obtained by local alignment of the double sequences is less than the 1 st threshold, and the global alignment score obtained by global alignment of the base sequences including the 3' -end of the base sequences of the primer candidates by the number of bases set in advance is less than the 2 nd threshold.
For example, it is conceivable to use the nucleotide sequences of SEQ ID Nos. 1 and 2 shown in Table 7 as the nucleotide sequences of primers for amplifying the target region.
[ Table 7]
TABLE 7
Figure BDA0001420562140000191
As already noted, the local alignment score is "-3", less than "3" as the 1 st threshold. And, the global alignment score is "-3", less than "3" as the 2 nd threshold.
Therefore, the nucleotide sequence of the primer candidate represented by SEQ ID No. 1 and the nucleotide sequence of the primer candidate represented by SEQ ID No. 2 can be used as the nucleotide sequences of the primers for amplifying the target region.
[ method for designing primer for polymerase chain reaction (embodiment 2) ]
The method for designing a primer for polymerase chain reaction according to embodiment 2 of the present invention comprises the following steps.
(a1) 1 st target region selection step of selecting a 1 st target region amplified by polymerase chain reaction from a region on the genome;
(b1) A 1 st primer candidate base sequence preparing step of preparing at least 1 base sequence of each primer candidate for amplifying the 1 st target region, based on the base sequences of the respective vicinal regions at both ends of the 1 st target region on the genome;
(c1) A 1 st local alignment step of obtaining a local alignment score by performing local alignment of double sequences on the base sequences of the primer candidates under such a condition that a part of the sequences to be compared include the 3' -end of the base sequence of the primer candidate;
(d1) The first stage 1 of the selecting step according to the above (c)1) 1, performing stage 1 selection of the candidate base sequences of the primers based on the local alignment scores obtained in the local alignment step;
(e1) 1 the global alignment step of preliminarily aligning the 3' -end of the nucleotide sequence including the candidate primerGlobal alignment is carried out on the base sequences with the set sequence length by double sequences, so as to obtain global alignment scores;
(f1) The first 2 nd stage selecting step according to the step (e)1) The global alignment score obtained in the 1 st global alignment step is used for the 2 nd stage selection of the candidate base sequences of the primers;
(g1) Step 1 of using the primer described in (d)1) A first stage 1 selecting step and (f) above1) The nucleotide sequence of the primer candidate selected in any of the first 2 nd step selection step is used as the nucleotide sequence of the primer for amplifying the 1 st target region,
and further comprises:
(an) An nth target region selection step of selecting an nth target region amplified by polymerase chain reaction from a region on the genome;
(bn) An nth primer candidate base sequence creating step of creating at least 1 base sequence of each of primer candidates for amplifying the nth target region on the basis of the base sequence of each of the regions in the vicinity of both ends of the nth target region on the genome;
(cn) An nth partial alignment step of obtaining a partial alignment score by partially aligning the nucleotide sequence of the primer candidate for amplifying the nth target region and the nucleotide sequence of the used primer in a double sequence under a condition that a part of the sequences to be compared include the nucleotide sequence of the primer candidate and the 3' -end of the nucleotide sequence of the used primer;
(dn) The nth stage 1 selection step is based on the step (c)n) Selecting the primer candidate base sequence for amplifying the nth target region at stage 1 based on the local alignment score obtained in the nth local alignment step;
(en) An nth global alignment step of aligning nucleotide sequences of predetermined sequence lengths including the nucleotide sequence of the primer candidate for amplifying the nth target region and the 3' -end of the nucleotide sequence of the used primer in a double sequencePerforming global alignment on the columns so as to obtain global alignment scores;
(fn) The nth 2 nd stage selection step according to the above (e)n) Selecting at stage 2 a candidate base sequence of a primer for amplifying the nth target region based on the global alignment score obtained in the nth global alignment step; and
(gn) The nth primer used in the step (d)n) (n) the 1 st stage of the selection step and (f) aboven) The nucleotide sequence of the candidate primer selected in any of the n-2 nd step is used as the nucleotide sequence of the primer for amplifying the n-th target region.
Wherein n is an integer of 2 or more, and the above (a) is repeated for all target regions until n reaches the number of target regions selected in the target region selection stepn) (n) target region selection step to (g) aboven) The nth primer is used in the respective steps until the base sequence of the primer for amplifying the target region is used.
Wherein (c) above1) 1 st local alignment step and the above (d)1) The two steps of the first stage 1 selection step may be as described above (e)1) 1 st Global alignment Process and (f) above1) Before or after the two steps of the first 2 nd stage selection step, or the step (e)1) 1 st Global alignment Process and (f) above1) The first 2 nd stage selecting step, which is performed simultaneously, and the step (c) described aboven) (n) th local alignment step and (d) aboven) The two steps of the n-th stage 1 selection step may be as described above (e)n) (n) th global alignment step and (f) aboven) Before or after the two steps of the n-th 2 nd stage selection step, or the same as (e)n) (n) th global alignment step and (f) aboven) The two processes of the nth 2 nd stage selection process are simultaneously carried out.
The respective steps of embodiment 2 of the method for designing a primer of the present invention will be described in detail.
(a1) 1 st target region selection step
Shown in the block diagram of fig. 1 as "(1 st target region selection process").
The same procedure as in the "target region selection step (a)" of embodiment 1 is repeated except that 1 gene region is selected as the 1 st target region from the regions on the genome.
(b1) Process for preparing candidate nucleotide sequence of primer 1
Shown in the block diagram of FIG. 1 are "(1 st step of preparing a candidate base sequence for a primer").
Prepared for amplification in (a) above1) The base sequence of the primer candidate for the 1 st target region selected in the 1 st target region selection step is the same as the "base sequence candidate for primer" in the 1 st aspect of the design method of the present invention except that the above steps are repeated.
(characteristic inspection step)
The same as the "characteristic inspection step" of the first aspect of the design method of the present invention. This step may be carried out or not.
(c1) 1 st local alignment step
Shown in the block diagram of fig. 1 as "(1 st partial alignment step").
To the above (b)1) The same procedure as the "local alignment step (c)" of the 1 st aspect of the design method of the present invention is repeated except that the base sequences of the primer candidates for amplifying the 1 st target region prepared in the 1 st primer candidate base sequence preparation step are locally aligned.
(d1) First stage 1 selecting process
Shown in the block diagram of fig. 1 as "(first) stage 1 election process".
According to the above (c)1) The local alignment score obtained in the 1 st local alignment step is calculated in the above (b)1) The method is the same as the "selection step at stage 1" in the 1 st embodiment of the design method of the present invention except that the nucleotide sequence of the primer candidate for amplifying the 1 st target region prepared in the 1 st primer candidate nucleotide sequence preparation step is selected as the target.
(e1) 1 st Global alignment procedure
Shown in the block diagram of fig. 1 as "(1 st) global alignment procedure".
To the above (b)1) The same procedure as the "global alignment step" as defined in the 1 st embodiment of the design method of the present invention is repeated except that the base sequences of the primer candidates for amplifying the 1 st target region prepared in the 1 st primer candidate base sequence preparation step are globally aligned.
(f1) First 2 nd stage selecting and drawing process
Shown in the block diagram of fig. 1 as "(first) stage 2 election process".
According to the above (e)1) The global alignment score obtained in the 1 st global alignment step is calculated in the above (b)1) The method is the same as the "2 nd stage selection step" in the 1 st embodiment of the design method of the present invention except that the nucleotide sequence of the primer candidate for amplifying the 1 st target region prepared in the 1 st primer candidate nucleotide sequence preparation step is selected as the target.
(c) the same as in the 1 st embodiment of the designing method of the present invention, wherein1) 1 st local alignment step and the above (d)1) The two steps of the first stage 1 selection step may be as described above (e)1) 1 st Global alignment Process and (f) above1) The two steps of the first 2 nd stage selection step may be performed before or after (e)1) 1 st Global alignment Process and (f)1) The first 2 nd stage selection step is performed simultaneously.
Further, as in the case of the 1 st embodiment, it is preferable to carry out "(e) first in order to reduce the amount of calculation1) 1 st Global alignment Process "and" (f)1) The first 2 nd stage selecting step ", the combination of the first 2 nd stage selecting step is performed" (c)1) 1 th local alignment Process "and" (d)1) First stage 1 selection process ".
(step of examining the length of amplification sequence)
This is the same as the "inspection procedure for the length of amplified sequence" in the 1 st embodiment. This step may be carried out or not.
(g1) Step 1 of primer application
Shown in the block diagram of FIG. 1 as "(1 st primer application step").
From the above (b)1) The same procedure as the "(g) primer application procedure" of embodiment 1 of the design method of the present invention is repeated except that the nucleotide sequence of the primer candidate for amplifying the 1 st target region prepared in the 1 st primer candidate nucleotide sequence preparation procedure is used.
In embodiment 2 of the present invention, after designing a primer for amplifying the 1 st target region, a primer for amplifying the nth (n is an integer of 2 or more) target region is designed.
(an) N-th target region selection step
The block diagram of fig. 1 is shown as "nth target region selection process".
The method is the same as the "(a) target region selection step" of embodiment 1 except that 1 gene region is selected as the nth target region from among the regions on the genome up to the region that has not been selected in the (n-1) th target region selection step.
In addition, the selection of the nth target region can be performed simultaneously with or after the selection of the (n-1) th target region. Wherein n is an integer of 2 or more.
(bn) Process for preparing n-th primer candidate base sequence
The block diagram of FIG. 1 shows "the nth primer candidate base sequence preparation step".
Prepared for amplification in (a) aboven) The base sequence of the primer candidate for the nth target region selected in the nth target region selection step is the same as the "base sequence candidate for primer" in the 1 st aspect of the design method of the present invention except that the above steps are repeated.
(characteristic inspection step)
The same as the "characteristic inspection step" of the first aspect of the design method of the present invention. This step may be carried out or not.
(cn) N-th local alignment step
The block diagram of fig. 1 is shown as "nth partial alignment process".
To the above (b)n) N primerThe "local alignment step (c)" of the embodiment 1 of the present invention is the same except that the nucleotide sequence of the candidate primer for amplifying the nth target region created in the candidate nucleotide sequence creating step and the nucleotide sequence of the used primer are locally aligned.
The base sequences of the primers used are all the base sequences (the same below) used as the base sequences of the primers for amplifying the 1 st to (n-1) th target regions.
(dn) N 1 st stage selecting and drawing process
The block diagram of fig. 1 is shown as "nth stage 1 election process".
According to the above (c)n) The local alignment score obtained in the n-th local alignment step is calculated in the step (b)n) The method of designing a target region of the present invention is the same as the "selection step at stage 1" (d) of the first embodiment "of the present invention except that the nucleotide sequence of the primer candidate for amplifying the nth target region prepared in the nucleotide sequence candidate preparing step and the nucleotide sequence of the primer used are selected.
(en) N-th global alignment procedure
Shown in the block diagram of fig. 1 as "nth global alignment procedure".
To the above (b)n) The "global alignment step" of embodiment 1 of the present invention is the same as the "global alignment step" except that the nucleotide sequence of the primer candidate for amplifying the nth target region created in the nth primer candidate nucleotide sequence creating step and the nucleotide sequence of the primer used are globally aligned with each other.
(fn) N 2 nd stage selecting and drawing process
The block diagram of fig. 1 is shown as "nth stage 2 election process".
According to the above (e)n) The global alignment score obtained in the n-th global alignment step is calculated in the above (b)n) The nucleotide sequence of the primer candidate for amplifying the nth target region prepared in the nth primer candidate nucleotide sequence preparation step and the nucleotide sequence of the used primer are selected and dividedThe procedure is otherwise the same as in "(f) 2 nd stage selection step" of the 1 st aspect of the design method of the present invention.
In addition, the above (c) is the same as the 1 st aspect of the design method of the present inventionn) (n) th local alignment step and (d) aboven) The two steps of the n-th stage 1 selection step may be as described above (e)n) (n) th global alignment step and (f) aboven) The n-th 2 nd stage selection step may be carried out before or after the two steps, and may be carried out in the same manner as (e)n) N-th global alignment step and (f)n) The two processes of the nth 2 nd stage selection process are simultaneously carried out.
In order to reduce the amount of calculation, it is preferable to first perform the above (e)n) (n) th global alignment step and (f) aboven) The two steps of the n 2 nd stage selection step pass through the step (f)n) (n 2 nd stage of the selection step in combination with the (c)n) (n) th local alignment step and (d) aboven) Two steps of the nth stage 1 selecting step. In particular, the effect of reducing the amount of calculation increases as the number of target regions increases and the number of base sequences of primer candidates increases, and the entire process can be speeded up.
(step of examining the length of amplification sequence)
This is the same as the "inspection step for the length of amplified sequence" in the 1 st embodiment of the design method of the present invention. This step may be performed or not performed as an arbitrary step.
(gn) The nth primer adopts the procedure
The block diagram of FIG. 1 shows "the nth primer application step".
From the above (b)n) The same procedure as the "(g) primer application procedure" of embodiment 1 of the design method of the present invention is repeated except that the nucleotide sequence of the primer candidate for amplifying the nth target region prepared in the nth primer candidate nucleotide sequence preparation procedure is used.
[ primer combination for polymerase chain reaction ]
The primer set for polymerase chain reaction of the present invention is a primer set designed by the above-described method for designing a primer for polymerase chain reaction.
That is, the local alignment score obtained by locally aligning the base sequences of the primers in a double sequence under the condition that a part of the sequences to be compared include the 3 '-end of the base sequences is less than the 1 st threshold, and the global alignment score obtained by globally aligning the base sequences including the 3' -end of the base sequences of the primers in a double sequence in a predetermined number of bases is less than the 2 nd threshold.
In addition, the global alignment score obtained by globally aligning base sequences of a predetermined number of bases including the 3 '-end of the base sequence of each primer in a double sequence can be made smaller than the 2 nd threshold by setting the base sequences of several bases up to the 3' -end of the primer as the same base sequence.
By using the present invention, SNP determination and/or SNV determination can be performed for a very small amount of DNA. Furthermore, by setting the target region at an arbitrary position within the specific chromosome, the quantitative ratio of each specific chromosome can be known. This enables the detection of genomic abnormalities from single cells or very small amounts of DNA, such as prenatal diagnosis.
The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to these examples.
Examples
[ example 1]
(1) Selection of target area
SNP positions shown in Table 8 were selected as target regions. Further, SNP D is an identification number used in dbSNP (Single Nucleotide Polymorphism Database) managed by NCBI (National Center for Biotechnology Information: National Center for Biotechnology, USA).
[ Table 8]
TABLE 8
SNP ID Chromosome numbering Coordinates of the object Allyl radical
rs7981616 13 25265103 A/G
rs2230233 18 29104698 C/T
rs2073370 21 35260481 T/C
rs2379206 X 6995315 C/T
(2) Preparation of candidate base sequences for primers
For each of the selected target regions, base sequences of primer candidates shown in Table 9 (SEQ ID Nos. 1 to 8) were prepared. Here, the "forward primer" refers to a primer prepared from the base sequence of the region 5 'to the SNP site (region on the smaller coordinate side), and the "reverse primer" refers to a primer prepared from the base sequence of the region 3' to the SNP site (region on the larger coordinate side) (base sequence of the complementary strand of chromosomal DNA).
[ Table 9]
TABLE 9
Figure BDA0001420562140000261
(3) Evaluation of dimer Forming Properties
In order to exclude a primer candidate base sequence which is likely to form a primer dimer, dimer formation is evaluated by local alignment score, and a primer candidate base sequence which is evaluated as having low dimer formation is further evaluated by global alignment score, thereby obtaining a combination of primer candidate base sequences having low dimer formation.
a) Evaluation based on local alignment scoring
For all pairs (28 sets) of the primer candidate base sequences represented by SEQ ID Nos. 1 to 8, the double sequences were partially aligned under the condition (constraint condition) that a part of the base sequences to be compared included the 3' -end of the primer candidate base sequence. For the local alignment, the alignment score was maximized under the above constraint conditions by using the scoring system shown in table 10. In table 10, "-" indicates a gap (indel, insertion/deletion).
The threshold for the local alignment score was set to 3, with pairs less than 3 passing.
[ Table 10]
Watch 10
Figure BDA0001420562140000271
The resulting alignment is shown in the graphs of fig. 2 to 29. For all combinations, the local alignment score was less than "3" and evaluated as less dimer formation.
b) Evaluation based on global alignment scoring
For all pairs (28 groups) of the primer candidate base sequences represented by SEQ ID Nos. 1 to 8, 3 bases from the 3 ' -end of the primer candidate base sequence were extracted, and base sequences (all 5 ' -TGG-3 ') including the 3 bases were globally aligned. For global alignment, the alignment score was maximized using the scoring matrix shown in table 10. The threshold for the score was set to 3, with pairs less than 3 passing. In table 10, "-" indicates a gap (indel, insertion/deletion).
The obtained alignment is shown in fig. 30. For all pairs, the global alignment score was less than "3" and evaluated as less dimer formation.
(4) Evaluation of distance between primers
Based on the combination of the primer candidate base sequences with low dimer formation obtained by the above "(3) evaluation of dimer formation", the inter-primer distance (length from the amplification start position to the amplification end position) was calculated for each of the pairs of primer candidates (the pair of sequence numbers 1 and 2, the pair of sequence numbers 3 and 4, the pair of sequence numbers 5 and 6, and the pair of sequence numbers 7 and 8) for PCR amplification of each target region.
Pairs with a distance between the primers in the range of 160 to 180 bases were passed.
As shown in Table 9, the inter-primer distances of all the primer candidate pairs were in the range of 160 to 180 bases.
(5) Determination of primer combinations
As a primer set for amplifying SNP positions at 4 positions shown in Table 1 by multiplex PCR, a primer set (primer set) including base sequences represented by SEQ ID Nos. 1 to 8 was obtained.
The primer combination enables simultaneous selective and efficient amplification of a target region by multiplex PCR even when a very small amount of genomic DNA extracted from a single cell with low primer dimer formation is used as a template DNA.
Comparative example 1
(1) Preparation of candidate base sequences for primers
As the primer candidate nucleotide sequences, a pair of nucleotide sequences represented by SEQ ID No. 9 and nucleotide sequence represented by SEQ ID No. 10 was prepared.
(2) Evaluation of dimer Forming Properties
The threshold of the local alignment score was set to "3" and the pair having a higher dimer formation property than "3", and the dimer formation property was not evaluated and excluded.
The pair of nucleotide sequences represented by SEQ ID Nos. 9 and 10 was partially aligned in the same manner as in example 1.
The obtained alignment is shown in fig. 31. Since the partial alignment score of the pair of nucleotide sequences represented by SEQ ID Nos. 9 and 10 was "7" and was equal to or greater than "3" as a threshold, the pair was excluded as a pair having high dimer formation.
Further, when PCR (Polymerase Chain Reaction) is actually performed using the primer represented by the base sequence number 9 and the primer represented by the base sequence number 10, a primer dimer can be formed. Dimers obtained from such high-scoring alignments can be prevented by the stage 1 selection step.
[ Table 11]
TABLE 11
Figure BDA0001420562140000281
Comparative example 2
(1) Preparation of candidate base sequences for primers
As the primer candidate nucleotide sequences, a pair of the nucleotide sequence represented by SEQ ID No. 11 and the nucleotide sequence represented by SEQ ID No. 12 was prepared.
(2) Evaluation of dimer Forming Properties
a) Evaluation based on local alignment scoring
The threshold of the local alignment score was set to "3", and pairs smaller than "3" were passed, and dimer formation was further evaluated.
The pair of nucleotide sequences represented by SEQ ID Nos. 11 and 12 was partially aligned in the same manner as in example 1.
The alignment obtained is shown in figure 32. Since the pair of base sequences represented by SEQ ID Nos. 11 and 12 had a local alignment score of "-4" and was smaller than "3" as a threshold, the pair was further evaluated for dimer formation.
b) Evaluation based on global alignment scoring
The threshold of the global alignment score was set to "2" and the pair having a higher dimer formation property than "2" and was excluded without further evaluation of the primers.
The pair of nucleotide sequences represented by SEQ ID Nos. 11 and 12 was aligned globally in the same manner as in example 1.
The alignment obtained is shown in figure 33. Since the global alignment score for the 2 bases at the 3' -end of the pair of base sequences represented by SEQ ID Nos. 11 and 12 is "2" or more which is a threshold value, the pair is excluded as a pair having a high dimer formation property.
Thus, since a primer dimer is formed only by a few bases at the 3 'terminal part of the primer, the alignment score is low when viewed from the entire sequence by performing the 2 nd selection step in addition to the 1 st selection step, but dimer formation due to annealing by only a few bases at the 3' terminal part can be prevented.
Industrial applicability
The present invention can provide a primer set that can selectively and efficiently amplify a target region even when the number of amplified gene regions is relatively small or large, and is useful for PCR using a very small amount of genomic DNA (deoxyribose nucleic acid) as a template DNA, such as PCR (Polymerase Chain Reaction) amplification from a single cell, and thus can be applied to various applications using a PCR method, including gene diagnostic applications.
Sequence listing
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Claims (2)

1. A method for designing a primer for polymerase chain reaction, comprising:
a target region selection step of selecting a target region amplified by the polymerase chain reaction from a region on a genome;
a primer candidate base sequence creating step of creating at least 1 base sequence of a primer candidate for amplifying the target region, respectively, based on the base sequences of the regions in the vicinity of both ends of the target region on the genome;
a local alignment step of obtaining a local alignment score by locally aligning the base sequences of the primer candidates with a double sequence under such a condition that the compared partial sequence includes the 3' -end of the base sequence of the primer candidate;
a stage 1 selection step of performing stage 1 selection of the base sequence of the primer candidate based on the local alignment score obtained in the local alignment step;
a global alignment step of obtaining a global alignment score by globally aligning base sequences of a predetermined sequence length including the 3' -end of the base sequence of the primer candidate in a double sequence;
a stage 2 selection step of selecting stage 2 of the base sequence of the primer candidate based on the global alignment score obtained in the global alignment step; and
a primer-using step of using, as a base sequence of a primer for amplifying the target region, a base sequence of a candidate primer selected in either of the step 1 and the step 2,
wherein the two steps of the local alignment step and the stage 1 selection step are performed after the two steps of the global alignment step and the stage 2 selection step.
2. The method for designing a primer for polymerase chain reaction according to claim 1, comprising:
1 st target region selection step of selecting a 1 st target region amplified by the polymerase chain reaction from a region on a genome;
a 1 st primer candidate base sequence creating step of creating at least 1 base sequence of each of primer candidates for amplifying the 1 st target region, based on the base sequences of the regions in the vicinity of both ends of the 1 st target region on the genome;
a 1 st local alignment step of obtaining a local alignment score by performing local alignment of double sequences on the base sequences of the primer candidates under such a condition that a part of the sequences to be compared include the 3' -end of the base sequence of the primer candidate;
a first stage 1 selection step of performing stage 1 selection of the base sequence of the primer candidate based on the local alignment score obtained in the local alignment step;
a 1 st global alignment step of performing global alignment of base sequences of a predetermined sequence length including the 3' -end of the base sequence of the primer candidate in a double sequence to obtain a global alignment score;
a first stage 2 selection step of selecting stage 2 of the base sequence of the primer candidate based on the global alignment score obtained in the global alignment step;
a 1 st primer application step of applying, as a base sequence of a primer for amplifying the 1 st target region, a base sequence of a candidate primer selected in either of the first 1 st and 2 nd step;
a 2 nd target region selection step of selecting a 2 nd target region amplified by the polymerase chain reaction from a region on the genome;
a 2 nd primer candidate base sequence preparing step of preparing at least 1 base sequence of each of the primer candidates for amplifying the 2 nd target region, based on the base sequences of the regions in the vicinity of both ends of the 2 nd target region on the genome;
a 2 nd local alignment step of obtaining a local alignment score by locally aligning the nucleotide sequence of the primer candidate for amplifying the 2 nd target region and the nucleotide sequence of the used primer in a double sequence under a condition that a part of the sequences to be compared includes the nucleotide sequence of the primer candidate and the 3' -end of the nucleotide sequence of the used primer;
a second stage 1 selection step of performing stage 1 selection of a base sequence of a candidate primer for amplifying the 2 nd target region based on the local alignment score;
a 2 nd global alignment step of performing global alignment of base sequences of a predetermined sequence length including the base sequence of the primer candidate for amplifying the 2 nd target region and the 3' end of the base sequence of the used primer in a double sequence to obtain a global alignment score;
a second stage 2 selection step of performing stage 2 selection of a base sequence of a candidate primer for amplifying the 2 nd target region based on the global alignment score; and
a 2 nd primer-employing step of employing, as a base sequence of a primer for amplifying the 2 nd target region, a base sequence of a candidate primer selected in either of the second 1 st-stage selection step and the second 2 nd-stage selection step,
wherein the 1 st local alignment step and the first 1 st stage selection step are performed after the 1 st global alignment step and the first 2 nd stage selection step, and
the 2 nd local alignment step and the second 1 st stage selection step are performed after the 2 nd global alignment step and the second 2 nd stage selection step,
when the target region is 3 or more, the steps from the 2 nd target region selection step to the 2 nd primer application step are repeated for all target regions until the base sequence of the primer for amplifying the target region is applied.
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